CN110343640A - A kind of fermentation process of people's intestinal flora and application - Google Patents
A kind of fermentation process of people's intestinal flora and application Download PDFInfo
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Abstract
The present invention provides a kind of fermentation process of people's intestinal flora and applications, and described method includes following steps: a. screens donor: 12-14 years old boy, health, without major disease history, without obvious gastrointestinal symptom, any antibiotic, rule of life were not taken at least six moon；B. sample collection and processing: acquiring the fresh excreta of donor, is transferred quickly to anaerobism work station, weighing, and the sterile saline of prereduction is proportionally added, and sufficient vortex mixes；Then it filters, collects excrement suspension；C. it ferments: excrement suspension being transferred to sterile serum bottle, sealing, which is placed in incubator, to be left to ferment.The pH that the present invention obtains acid and rich in a variety of probiotics tunning is a kind of intestinal flora of the Healthy People of optimization, can be used for caprophyl implantation technique and using intestinal flora as the exploitation of the drug of target spot, health care product etc..
Description
Technical field
The present invention relates to microorganism, clinic, drug and field of health care products, and in particular to a kind of people's intestinal flora fermentation process
And its application.
Background technique
Substantial amounts, miscellaneous microorganism are colonized in human body intestinal canal, the food digestion of their wide participation human bodies,
Equal physiological activities are metabolized and are immunized, it is closely bound up with human health.In recent years a large number of studies show that, intestinal flora is unbalance with it is fertile
The high-incidence chronic disease of the various moderns such as fat, diabetes, inflammatory bowel disease, colorectal cancer, self-closing disease society is closely related, adjusts
Intestinal microflora can play preventive and therapeutic action to disease.
The currently used method for adjusting intestinal flora includes taking probiotics or prebiotic substance, Dietary frequency, caprophyl
Transplanting etc..Wherein, it takes probiotics drug or health care product is using wide one kind, this method is by one plant or several of supplement
Strain generally acknowledged beneficial bacteria plays its probiotic effects.But there is studies have shown that single or the probiotics of limited kinds is in enteron aisle
In colonization ability it is poor, cannot effectively adjust the overall structure of host intestine flora and play its effect.Intestinal flora is one
The ecosystem of a complexity, bacterium therein are not independent, but " the function of being formed in ecology that interacts
Group " functions, to cope with the variation of system environments.Therefore, it is micro- to establish the high and enteron aisle rich in probiotics of a diversity
Biological function group is to realize more reasonable, the effective method for adjusting intestinal flora.
Caprophyl transplanting is a hot spot technology of medicine and microorganism group field in recent years, and this method has used Tiny ecosystem
The entire faecal microbiota of Healthy People is transferred in the enteron aisle of patient by concept, by changing the intestinal flora of patient, is improved
Enteron aisle stable state achievees the effect that treat disease.Researcher attempted by caprophyl transplant be introduced into recurrent clostridium difficile infection,
In the treatment of a variety of diseases such as inflammatory bowel disease, self-closing disease, but the effect treated is not consistent in different clinical researches.Transplanting
Intestinal flora composition in object is the key factor for determining caprophyl transplanting curative effect, but donor is different, excrement suspension preparation method is different
The flora in graft will be caused to form different.Some apparent its intestinal flora of the donor of " health " compositions are possible to not
" health " and balance, in some instances it may even be possible to which there are some potential opportunists, if transplanting such flora to patient, so that it may
The effect for the treatment of can be seriously affected.Therefore, development can optimize the method for people's intestinal microflora obtain prebiotic bacterial content it is high and
Intestinal flora composition without obvious pathogen has in terms of the development of caprophyl implantation technique and the exploitation of probiotic products and answers
Use prospect.
Temperature is to influence a key factor of bacterial growth.Existing intestinal flora fermentation mostly uses 37 DEG C, there is research
Display carries out the in vitro culture and fermentation of intestinal flora, Proteobacteria (Proteobacteria) bacterial content at such a temperature
It dramatically increases, and in this includes a variety of generally acknowledged pathogens and common opportunist.Therefore, intestines under lower temperature are explored
The feature that bacterium forms after road microflora fermentation, it is possible to it is high to establish a kind of low and potential prebiotic bacterial content of opportunistic diseases bacterial content
The healthier intestinal flora of structure.
Summary of the invention
In view of the deficiencies of the prior art, the purpose of the present invention is to provide a kind of fermentation process of people's intestinal flora, the party
Method is simple and easy, strong operability.
The purpose of the present invention is what is be achieved through the following technical solutions:
The present invention provides a kind of fermentation process of people's intestinal flora, carry out in accordance with the following steps:
A. donor is screened: 12-14 years old boy, health, no major disease history, without obvious gastrointestinal symptom, at least six
Any antibiotic, rule of life were not taken in month；
B. sample collection and processing: acquiring the fresh excreta of donor, is transferred quickly to anaerobism work station, weighing, according to than
The sterile saline of prereduction is added in example, and sufficient vortex mixes；Then it filters, collects excrement suspension；
C. it ferments: excrement suspension being transferred to sterile serum bottle, sealing, which is placed in 10~20 DEG C of incubators, to be left to ferment.
Preferably, the sterile saline of the prereduction is that 0.9% sodium chloride solution divides through 121 DEG C of high-temperature sterilizations 20
Clock, the degassing of anaerobism work station and substitution gas handle 12 hours it is made above and obtain；The fresh excreta and prereduction it is sterile
The mixed proportion of physiological saline is 1:10 (w/v).
Preferably, in the fermentation step, the excrement suspension of use is the excrement suspension or 2~5 donors of 1 donor
Excrement suspension isometric mixture.
Preferably, the time that the excrement suspension is left to ferment in 10 DEG C of incubators is 30~120 days.
It is highly preferred that the time of the fermentation is 30~90 days.
The present invention also provides a kind of people's intestinal floras obtained according to aforesaid fermentation method in caprophyl implantation technique and
Using intestinal flora as the application in the drug of target spot, health care product.
Preferably, described in the application of caprophyl implantation technique, the people's intestinal flora for acquisition of fermenting is with excrement suspension or freeze-drying
The form application of microbial inoculum.
Preferably, it is described using intestinal flora as the drug of target spot, health care product application in, the people's intestinal flora for acquisition of fermenting
It is applied as probiotics, application forms are capsule, oral solution, tablet or electuary.
Preferably, the people's intestinal flora application in preparing anti-inflammatory drugs obtained including fermentation.
The present invention also provides a kind of people's intestinal floras obtained according to aforesaid fermentation method to inhibit proinflammatory cytokine
Application in IL-8 level.
People's intestinal flora fermentation process of the invention, by screening 12~14 years old virgin boy as donor and to excrement suspension into
Row low-temp anaerobic fermentation, obtained tunning have the advantages that:
(1) pH is reduced to 4.7~5.4, and the pH environment of this acidity can effectively inhibit a variety of opportunistic cause of diseases in enteron aisle
The growth of bacterium；
(2) tunning be one balance the intestinal flora ecosystem, wherein Lactobacillus (lactobacillus) and
The a variety of short chain fatty acids such as Blautia, Clostridium IV generate bacterial contents and dramatically increase, and Escherichia coli, salmonella,
The chances cause of disease bacterial content such as Shigella, Klebsiella is lower than 1% or can't detect；
(3) tunning has anti-inflammatory activity.
The intestinal flora tunning that the technical method of these behavioral illustrations through the invention obtains is a kind of people of optimization
Intestinal flora system.
Detailed description of the invention
Upon reading the detailed description of non-limiting embodiments with reference to the following drawings, other feature of the invention,
Objects and advantages will become more apparent upon:
Fig. 1 is the pH measurement in 1 fermentation process of embodiment；
Fig. 2 is the composition of bacterium in 1 fermentation system of embodiment；
Fig. 3 is the content of the OTUs (activity classification unit) of significant changes in 1 fermentation system of embodiment；
Fig. 4 is that the anti-inflammatory activity of 1 tunning of embodiment detects.
Specific embodiment
The present invention is described in detail combined with specific embodiments below.Following embodiment will be helpful to the technology of this field
Personnel further understand the present invention, but the invention is not limited in any way.It should be pointed out that the ordinary skill of this field
For personnel, without departing from the inventive concept of the premise, various modifications and improvements can be made.These belong to the present invention
Protection scope.
Embodiment 1:
A kind of fermentation process of people's intestinal flora, comprising the following steps:
(1) donor is screened: 3 12-14 years old boys of selection (2 12 years old, 1 13 years old), health, no major disease
History did not took any antibiotic, rule of life without obvious gastrointestinal symptom at least six moon.
(2) sample collection and processing: acquiring the fresh excreta of each donor, is transferred quickly to anaerobism work station, weighing,
The sterile saline of prereduction is added according to the ratio of 1:10 (w/v), sufficient vortex mixes；It is filtered, is collected with double gauze
Excrement suspension；
The sterile saline of the prereduction is 0.9% sodium chloride solution through 121 DEG C of high-temperature sterilizations 20 minutes, anaerobism work
Make station degassing and substitution gas handle 12 hours it is made above and obtain.
(3) it ferments: respectively taking the excrement suspension 150mL of 3 donors, be uniformly mixed, packing to sterile serum bottle, every bottle of 75mL,
It is sealed with silica gel plug and aluminium lid.Wherein 3 bottles are example 1 group, are placed in 10 DEG C of incubators and are left to ferment；Other 3 bottles are comparison
Example group, is placed in 37 DEG C of (Conventional bacteria fermentation temperature) incubators and is left to ferment.
Above-mentioned excrement suspension is left to ferment 120 days, and during which continuous acquisition sample carries out the analysis of pH and Bacterial community.
Example 1 group and comparative example group 2 days pH before fermentation are remarkably decreased, and are stablized after comparative example group 2 days on 5.6 left sides
The right side, and be still gradually reduced after example 1 group 2 days, pH is between 4.7~5.4.The result is shown in Figure 1.
In fermentation process, example 1 group is different with the changing rule of flora in comparative example group, in example 1 group,
The content of the bacterium of the categories such as Blautia, Roseburia, Clostridium IV, Lactobacillus is significantly higher than comparative example
Group, and the bacterial content of the categories such as Bacteroides, Ruminococcus, Romboutsia is higher than embodiment 1 in comparative example group
Group.In example 1 group, Shigella (Shigella), Klebsiella (Klebsiella), Enterococcus (intestines ball
Bacterium) etc. chances cause of disease bacterial content lower than 1% or can't detect.As a result see Fig. 2.
Fig. 3 is some OTUs for belonging to potential probiotics in two systems of example 1 group and comparative example group before and after fermentation
Changes of contents.In example 1 group, belong to Blautia, Roseburia, Coprococcus, Lactobacillus and
The abundance of multiple OTUs of Clostridium IV dramatically increases after fermentation, they are generally acknowledged at present Lactic Acid Producing and fourth
Hydrochlorate producing strains have potential probiotic effects.And in comparative example group, belong to Blautia, Faecalibacterium,
Multiple OTUs contents of Roseburia, Bifidobacterium, Anaerostipes, Butyricicoccus are remarkably decreased,
Even close to 0 after fermentation 90 days, show that potential probiotics significantly reduces after fermentation in the system.Above data shows this hair
Bright fermentation process forms one group of structure more preferably intestinal flora.
Fig. 4 is that the tunning of example 1 group and comparative example group secretes the Caco-2 cellular inflammation model that TNF-α induces
The influence of proinflammatory cytokine IL-8, the results show that example 1 group make Caco-2 cell generate IL-8 level be substantially less than pair
Ratio group, and it is horizontal far below the IL-8 that TNF-α induction generates.This illustrates that the tunning of embodiment 1 has anti-inflammatory activity.
Embodiment 2:
A kind of fermentation process of people's intestinal flora, comprising the following steps:
(1) donor is screened: 3 12-14 years old boys of selection (2 12 years old, 1 13 years old), health, no major disease
History did not took any antibiotic, rule of life without obvious gastrointestinal symptom at least six moon.
(2) sample collection and processing: acquiring the fresh excreta of each donor, is transferred quickly to anaerobism work station, weighing,
The sterile saline of prereduction is added according to the ratio of 1:10 (w/v), sufficient vortex mixes；It is filtered, is collected with double gauze
Excrement suspension；
The sterile saline of the prereduction is 0.9% sodium chloride solution through 121 DEG C of high-temperature sterilizations 20 minutes, anaerobism work
Make station degassing and substitution gas handle 12 hours it is made above and obtain.
(3) it ferments: respectively taking the excrement suspension 150mL of 3 donors, be uniformly mixed, packing to sterile serum bottle, every bottle of 75mL,
It is sealed with silica gel plug and aluminium lid.Wherein 3 bottles are 2 groups of embodiment, are placed in 20 DEG C of incubators and are left to ferment；Other 3 bottles are comparison
Example group, is placed in 37 DEG C of (Conventional bacteria fermentation temperature) incubators and is left to ferment.
Above-mentioned excrement suspension is left to ferment 120 days, and during which continuous acquisition sample carries out the analysis of pH and Bacterial community.
2 groups of embodiment and comparative example group 2 days pH before fermentation are remarkably decreased, and are stablized after comparative example group 2 days on 5.6 left sides
The right side, and pH is gradually stable after 4 days for 2 groups of embodiment, maintains 5.2 or so.Both comparisons intestinal flora composition, 2 groups of embodiment
Contained 24.53% Faecalibacterium and 18.55% Blautia at 120 days, it is (right to be significantly higher than comparative example group
Ratio group contains 5.30% Faecalibacterium and 0.59% Blautia).Faecalibacterium and
The bacterium that Blautia belongs to has stronger generation short chain fatty acids ability, is generally acknowledged beneficial bacteria of intestinal tract.In 2 groups of embodiment,
The chances pathogens such as Shigella (Shigella), Klebsiella (Klebsiella), Enterococcus (enterococcus) contain
Amount is not detected.Above data shows that the fermentation process of the present embodiment forms one group of structure more preferably intestinal flora.
Embodiment 3:
A kind of fermentation process of people's intestinal flora, comprising the following steps:
(1) donor is screened: 1 12 years old boy of selection, health, no major disease history, without obvious gastrointestinal symptom, until
Any antibiotic, rule of life were not taken in 6 months few.
(2) sample collection and processing: acquiring the fresh excreta of the donor, is transferred quickly to anaerobism work station, weighing, according to
The sterile saline of prereduction is added in the ratio of 1:10 (w/v), and sufficient vortex mixes；It is filtered with double gauze, it is outstanding to collect excrement
Liquid；
The sterile saline of the prereduction is 0.9% sodium chloride solution through 121 DEG C of high-temperature sterilizations 20 minutes, anaerobism work
Make station degassing and substitution gas handle 12 hours it is made above and obtain.
(3) ferment: taking the excrement suspension 225mL of the donor, packing to 3 sterile serum bottles, every bottle of 75mL, with silica gel plug and
Aluminium lid sealing.It is placed in 10 DEG C of incubators and is left to ferment 33 days, during which continuous acquisition sample carries out the analysis of Bacterial community.
When fermenting 33 days, pH falls to 5.35 for 3 groups of embodiment, belong to potential probiotics Bifidobaterium,
The bacterial content of the categories such as Blautia and Butyricicoccus is significantly higher than comparative example group, and common opportunistic pathogenic bacteria are basic
It can't detect.Fermentation process of the invention can obtain one group of people's intestinal flora optimized.
There are many concrete application approach of the present invention, the above is only a preferred embodiment of the present invention.More than it should be pointed out that
Embodiment is merely to illustrate the present invention, and the protection scope being not intended to restrict the invention.For the common skill of the art
For art personnel, without departing from the principle of the present invention, several improvement can also be made, these improvement also should be regarded as this hair
Bright protection scope.
Claims (11)
1. a kind of fermentation process of people's intestinal flora, which is characterized in that carry out in accordance with the following steps:
A. donor is screened: 12-14 years old boy, health, no major disease history, without obvious gastrointestinal symptom, at least six moon
Any antibiotic, rule of life were not taken；
B. sample collection and processing: acquiring the fresh excreta of donor, is transferred quickly to anaerobism work station, weighing, proportionally plus
Enter the sterile saline of prereduction, sufficient vortex mixes；Then it filters, collects excrement suspension；
C. it ferments: excrement suspension being transferred to sterile serum bottle, sealing, which is placed in incubator, to be left to ferment.
2. the fermentation process of people's intestinal flora according to claim 1, which is characterized in that the sterile physiological of the prereduction
Salt water is 0.9% sodium chloride solution to be handled 12 hours through 121 DEG C of high-temperature sterilizations 20 minutes, the degassing of anaerobism work station and substitution gas
It is made above and obtain；The mixed proportion of the fresh excreta and the sterile saline of prereduction is 1:10 (w/v).
3. the fermentation process of people's intestinal flora according to claim 1, which is characterized in that in the fermentation step, use
Excrement suspension be the excrement suspension of 1 donor or the excrement suspension of 2~5 donors isometric mixture.
4. the fermentation process of people's intestinal flora according to claim 1, which is characterized in that the fermentation temperature of the excrement suspension
It is 10 DEG C~20 DEG C.
5. the fermentation process of people's intestinal flora according to claim 1, which is characterized in that the fermentation time of the excrement suspension
It is 30~120 days.
6. the fermentation process of people's intestinal flora according to claim 5, which is characterized in that the time of the fermentation be 30~
90 days.
7. a kind of people's intestinal flora that any one of according to claim 1~6 fermentation process obtains caprophyl implantation technique with
And using intestinal flora as the application in the drug of target spot, health care product.
8. application according to claim 7, which is characterized in that described in the application of caprophyl implantation technique, fermentation obtains
People's intestinal flora by excrement suspension or freeze-drying microbial inoculum in the form of application.
9. application according to claim 7, which is characterized in that described using intestinal flora as the drug of target spot, health care product
In, the people's intestinal flora for acquisition of fermenting is applied as probiotics, application forms be capsule, oral solution, tablet or
Electuary.
10. applying according to claim 7, which is characterized in that the people's intestinal flora obtained including fermentation is preparing anti-inflammatory agent
Application in object.
11. a kind of people's intestinal flora that any one of according to claim 1~6 fermentation process obtains inhibit proinflammatory cytokines because
Application in sub- IL-8 level.
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